Conclusions
The similarity in clinical presentation for both COVID-19 and Influenza makes it difficult to assess their impact on ICU admission and mortality. Our study highlights the importance of screening for co-infecting viruses in COVID-19 patients, given the high prevalence of Influenza viruses. The detection of co-infections in COVID-19 cases shows the importance of flu vaccination and warrants its increased coverage to reduce the hospitalization and associated mortality.
Keywords: SARS-CoV-2, COVID-19, Influenza A H1N1, co-infection, ICU, comorbidities, mortality.
1. Introduction
The newly emergent Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to circulate outside of Wuhan, China since December 2019, and now exported to different countries all over the world [1]. At the time of writing this report, there were nearly quarter of a million of Coronavirus Disease-19 (COVID-19) confirmed cases ranking Saudi Arabia as the 14th highest in the world [2]. Most hospitalized patients needed admission to intensive care unit (ICU) and mortality reaches up to 50% in some cases [3]. Until now, twenty-two studies have reported co-infection in COVID 19 and of these 16 have evidence of viral co-infection [4]. The prevalence of critical cases with viral co-infection has been reported up to 35% [5]. Early literature reported that 50% of the patients who died had coexisting bacterial infection [6]. This is higher than what was previously seen during influenza pandemic in 2009 when 25% of patients with influenza infection had secondary bacterial co-infection [7].
SARS-CoV-2 is a single stranded RNA Betacoronavirus and belongs to the corona virus family [8]. Phylogenetic analysis has revealed that SARS-CoV-2 is closely related to SARS-CoV-1 and genetically distinct from MERS-CoV [9]. SARS-CoV-2 utilizes ACE-2 receptors in the lower airways which are also cellular receptors for other viruses in this group i.e. SARS-CoV and MERS-CoV [10]. Despite similar expression of ACE-2 receptors in different organs of the body, the most affected site is the lung tissue [11]. Influenza strains also cause lung damage by ACE-2 receptor mediated effects [12]. On the other hand, since the ACE-2 receptor used by SARS-CoV-2 is an interferon‐stimulated gene, it was hypothesized that type I and III interferons produced after bacterial infection may facilitate SARS‐CoV‐2 attachment [13].
During pandemics, the detection of the novel virus may lead to underreporting of other pathogens that could be the etiological agent contributing to the disease severity. Indeed, during the influenza A (H1N1) pdm09 pandemic, 44.3% of patients had unreported respiratory viruses [14]. Earlier studies indicated that common viral co-infections reported in COVID-19 patients include Influenza viruses, RSV and adenovirus [5,15]. Bacterial co-infection is more frequent than viral co-infection and it is homogeneously distributed in mild, moderate or severe illness [16]. The commonly known COVID-19 co-infecting bacteria are Mycoplasma pneumoniae, Pseudomonas aeruginosa, Hemophilus influenzae and Chlamydia pneumoniae [17]. These findings clearly emphasize on the importance of screening for other clinically important co-circulating respiratory pathogens contributing to the etiology of the disease.
The novelty of SARS-CoV-2 and the complicity of profound etiology of co-infection urged for consideration of comorbidities. COVID-19 patients with an underlying condition such as hypertension, diabetes, chronic kidney disease, and heart failure have been associated with COVID-19 disease severity [18]. Cardiovascular disease has a strong association with COVID-19 pneumonia (14.4%) [7,18] and other common comorbidities found in patients with SARS-CoV-2 include hypertension (18.6%) and diabetes (11.9%) [19]. Comorbidities were also linked with increased hospitalization, prolonged stay in ICU, and mortality. Hypertension was more prevalent in severe cases (47%) compared to diabetes (24%) and Respiratory diseases (10%) among other underlying conditions [18].
In conclusion, an extensive evidence revealed that viral infections predispose patients to subsequent bacterial co‐infections [7]. This knowledge gap is puzzling as limited number of reports have described prevalence of bacterial and viral co-infections simultaneously. We hypothesized that undetected co-infections might have severe clinical implications associated with increased hospitalization, prolonged stay in ICU, and mortality. Therefore, our aim was, to investigate the presence of viral and bacterial co-infections in ICU and non-ICU COVID-19 patients.
2. Materials and Methods
2.1. Patients
Forty-eight extracted RNA samples were collected from COVID-19 positive patients, of which 14 were critical cases needing admission to the ICU, and 34 were mild cases. Nine patients died, (all were admitted to the ICU), and the rest survived. Thirteen patients were Saudi citizens and the rest were non Saudi (Table 1). Samples were collected from King Fahad Hospital, Medina, Saudi Arabia. This study was approved by the Institutional Review Board at King Fahad Medical City (IRB Log No. 20-160). Informed consent to participate was waived or not required since only remaining left‐over specimens were used for this study.
2.2. RNA Extraction and PCR
Nasopharyngeal swabs were collected from the patients and carried in a suitable fluid viral medium. The RNA extraction of SARS-CoV-2 was performed via a MagNA Pure 96 machine, using the MagNA Pure 96 DNA and Viral NA small volume kit, (Roche, Germany). The amplification RT-PCR was performed within a Roche LightCycler® 480 II instrument, using the RNA Process Control Kit Trial Pack (Roche, Germany) with an internal, positive, and negative controls.
2.3. Real time PCR panel for Co-infection
Eluted nucleic acid was stored at −80°C until use, and all reagents were stored at −20°C. The quantitative RT-PCR assay for respiratory pathogens was performed on 7500 Fast Real-Time PCR System (Thermo Fisher scientific, USA). Extracted nucleic acid was screened by RT/q-PCR with Fast Track Diagnostic (FTD) Respiratory pathogens 21 plus kit (Biomerieux, Luxemburg) following the manufacturer’s protocol using six multiplex PCR for respiratory viruses and bacteria. The pathogens tested were influenza A (H1N1) virus (swine-lineage); influenza B virus; human rhinovirus; human coronaviruses NL63, 229E, OC43 and HKU1; human parainfluenza viruses 1, 2, 3 and 4; human metapneumoviruses A/B; human bocavirus; human respiratory syncytial viruses A/B; human adenovirus; enterovirus; human parechovirus; Mycoplasma pneumoniae; Chlamydia pneumoniae; Staphylococcus aureus; Streptococcus pneumoniae; Haemophilus influenzae B. Six positive controls were performed with every run (five for each viral panel and one for bacterium) on multiplex PCR assay. Moreover, six negative controls (NC), provided in the kit, were incorporated with each run. Briefly, 10 µl of the extracted nucleic acid was used as a template in each reaction for the FTD Respiratory pathogens 21 plus multiplex PCR following the manufacturer’s instructions. The thermal cycle amplification condition includes reverse transcription for 15 minutes at 42°C, denaturation for 3 minutes at 94°C followed by 40 cycles for 8 seconds at 94°C, and 34 seconds at 60°C. Specimens were determined to be pathogen positive or negative based on the manufacturer’s interpretation criteria, and 12 samples were randomly chosen and repeated to confirm the results.
2.4. Data collection
Demographic and clinical data (Table 1) were collected, including the following clinical laboratory results: age, gender, history of chronic illness, Ct value, Dimar, CK, CK-MB, Trop, HB, Platalet, RBC, WBC, Nutrophile, Lymph, CRP, Pro calciponin, Glucose, ESR, LDL, AST, ALT, Ureae, Creatinine, LDH, Albumin, Total Protein and blood group.